Segmental isotopic labeling of the Hsp70 molecular chaperone DnaK using expressed protein ligation

Clérico, Eugenia M.; Zhuravleva, Anastasia; Smock, Robert G.; Gierasch, Lila M.

doi:10.1002/bip.21426

Cited by 15 publications

(13 citation statements)

References 51 publications

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“…In Vivo Assays-Phage propagation and heat shock assays were performed as described previously using E. coli strain BB1553 transformed with plasmid pMS119-dnaK (13,25). In the ⌬secB assay with controllable dnaK expression, colonies of E. coli strain GP502 transformed with pMS119-dnaK plasmids were used to inoculate overnight cultures grown at 30°C in LB media supplemented with 0.5% arabinose, 50 mg/liter ampicil- (6,29,58), the NBD (green) is joined to the SBD (blue) through a short interdomain linker (red).…”

Section: Methodsmentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

Journal of Biological Chemistry

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The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide-and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.The ubiquitously distributed Hsp70 family of molecular chaperones shepherd newly synthesized polypeptide chains, protect cells from stress-induced protein aggregation, assist in protein translocation across membranes, and regulate assembly and disassembly of macromolecular complexes. These physiological functions are accomplished by a two-domain allosteric mechanism in which cycles of ATP binding and hydrolysis in the N-terminal nucleotide-binding domain (NBD) 2 control the binding and release of hydrophobic polypeptide segments in the substrate-binding domain (SBD) (1-3). Although we have gained an increasingly detailed picture of this allosteric transition in Hsp70 chaperones and modes of substrate interaction (4 -7), it is striking that there is much less known about the function of the extreme C-terminal unstructured region (Fig. 1). Binding of the C-terminal region of mammalian Hsc70 to substrate was suggested in earlier work (8, 9), and more recently, the C-terminal segment of eukaryotic cytoplasmic Hsp70s was found to contain a conserved tetratricopeptide repeat (TPR) domain interaction motif that mediates binding with Chip, Hip, or Hop co-chaperones that facilitate the assembly of a variety of Hsp70 complexes (3). Even though bacteria lack homologs of these Hsp70-interacting TPR domain proteins, many, including the extensively characterized E. coli Hsp70 DnaK, have a disord...

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Section: Methodsmentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

Journal of Biological Chemistry

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“…Although further optimization of the transpeptidation reactions under denaturing conditions is necessary to achieve optimal performance, the transpeptidation system developed in this study is poised to solve the previously reported problem of domain region spatial unavailability, which may limit the coupling reactions in certain cases [19].…”

Section: Discussionmentioning

confidence: 99%

“…As this result may be explained by the improved spatial availability of the termini of the domains being ligated [19], the primary objective of our work was to achieve efficient srtA-driven protein transpeptidation under denaturing conditions through protein modification.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

Zhulenkovs¹,

Jaudzems²,

Zajakina³

et al. 2014

Biochemical Engineering Journal

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“…Plasmids encoding DnaK NBD(1-388) T199A, NBD(1-392)T199A, and NBD(1-393)T199A were prepared previously in our laboratory; they were expressed in E. coli and purified as described (3,37). 2 H-, 13 C-, and 15 N-labeled NMR samples were prepared using established protocols (38), as described previously (37) (see SI Text).…”

Section: Methodsmentioning

confidence: 99%

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

Zhuravleva

Gierasch

2011

Proc. Natl. Acad. Sci. U.S.A.

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134

198

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The 70-kDa heat shock protein (Hsp70) chaperones perform a wide array of cellular functions that all derive from the ability of their N-terminal nucleotide-binding domains (NBDs) to allosterically regulate the substrate affinity of their C-terminal substrate-binding domains in a nucleotide-dependent mechanism. To explore the structural origins of Hsp70 allostery, we performed NMR analysis on the NBD of DnaK, the Escherichia coli Hsp70, in six different states (ligand-bound or apo) and in two constructs, one that retains the conserved and functionally crucial portion of the interdomain linker (residues 389 VLLL 392 ) and another that lacks the linker. Chemical-shift perturbation patterns identify residues at subdomain interfaces that constitute allosteric networks and enable the NBD to act as a nucleotide-modulated switch. Nucleotide binding results in changes in subdomain orientations and long-range perturbations along subdomain interfaces. In particular, our findings provide structural details for a key mechanism of Hsp70 allostery, by which information is conveyed from the nucleotide-binding site to the interdomain linker. In the presence of ATP, the linker binds to the edge of the IIA β-sheet, which structurally connects the linker and the nucleotide-binding site. Thus, a pathway of allosteric communication leads from the NBD nucleotide-binding site to the substrate-binding domain via the interdomain linker. T he 70-kDa heat shock proteins (Hsp70s) compose one of the most well studied and ubiquitously distributed families of allosteric proteins (1). Hsp70s assist in an extraordinarily broad spectrum of cellular processes, including protein folding, disaggregation, and translocation. All chaperone activities of Hsp70s are based on their ability to interact with short hydrophobic peptide segments of protein substrate in an ATP-dependent fashion. Hsp70s contain two domains-a 44-kDa N-terminal nucleotidebinding domain (NBD) and a 15-kDa C-terminal substrate-binding domain (SBD)-connected by a highly conserved hydrophobic linker. The allosteric cycle of Hsp70s involves an alternation between the ATP-bound state with low affinity and fast exchange rates for substrates, and the ADP-bound state with high affinity and low exchange rates for substrates. In turn, substrate binding to the SBD results in about 10-fold stimulation of ATPase activity of the NBD. However, the same ATPase stimulation can be achieved for the isolated NBD in the presence of the conserved interdomain linker sequence motif ( 389 VLLL 392 ) (2-4), indicating that the linker plays a key role in NBD function and allostery.The Hsp70 NBD belongs to the Actin/Hexokinase/Hsp70 superfamily, members of which share a number of common features (5, 6). The NBD is composed of two lobes, I and II; each lobe consists, in turn, of two subdomains: IA and IB for lobe I, and IIA and IIB for lobe II. Nucleotide binds at the bottom of the deep central cleft at the interface between subdomains IB and IIB, and all four subdomains are involved in nucleotide coordination...

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Segmental isotopic labeling of the Hsp70 molecular chaperone DnaK using expressed protein ligation

Cited by 15 publications

References 51 publications

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

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